PROJECT SUMMARY Increasing evidence shows that a broad spectrum of both industrial and environmental chemicals can cause female ovarian toxicity (ovotoxicity) and increase the risk of hormonal imbalance, ovarian failure, and infertility in both humans and other mammalian species. The ovary is the primary female reproductive organ and functions to synthesize and secret steroid hormones and to support the oocyte maturation for ovulation, fertilization, and pregnancy. Ovotoxicity can be measured in two ways, from the health of the developing ovarian follicle and gamete and from the ability of the gonads to produce hormones. Unfortunately, the lack of optimal in vitro models makes the current gold standard for testing the ovotoxicity of chemicals rely on the use of laboratory animals. However, in vivo models are time-consuming, costly, and harmful to animals. In our previous studies, we have used an alginate hydrogel-based encapsulation method to grow both mouse and human preantral follicles outside of the context of the ovary, which is termed encapsulated in vitro follicle growth (eIVFG). The eIVFG maintains the 3D architecture of the follicles and supports follicle growth, hormone secretion as well as oocyte maturation and ovulation, suggesting a promising model for in vitro ovotoxicity testing. However, this method is relatively slow, and multiple rounds of follicle isolation, culture, and chemical exposure are necessary to complete dose-response and time-course experiments, making the current eIVFG scenario not optimal, particularly when a rapid screening is required in the case of unpredictable and emerging environmental threats. In this proposed project, we hypothesize that we can use cryopreservation technology and a confocal high-content imaging system to develop a high-throughput platform for screening ovotoxic chemicals. In Aim 1, we will bank the isolated ovarian follicles through vitrification for establishing a long-term and ready-to-use follicle storage platform. In Aim 2, we will use the ImageXpress confocal high-content imaging system to develop a high-throughput platform for follicle monitoring, assessing, and analysis. We expect that these proposed studies will allow us to obtain rapid and reliable evidences to understand the impacts of environmental contaminants on the female reproductive system, to provide guidance to conduct preventive and protective activities, as well as to minimize the dependence and use of animals.